Cell-type diversity and connectome of the ctenophore apical organ


Kei Jokura1,3,4,*, Sanja Jasek1,2, Lara Kewalow2, Pawel Burkhardt5, Gáspár Jékely1,2,*

1Living Systems Institute, University of Exeter, Exeter, EX4 4QD, United Kingdom
2Heidelberg University, Centre for Organismal Studies (COS), 69120 Heidelberg, Germany
3Exploratory Research Center on Life and Living Systems (ExCELLS), National Institutes of Natural Sciences, Okazaki, Japan
4National Institute for Basic Biology (NIBB), National Institutes of Natural Sciences, Okazaki, Japan
5Michael Sars Centre, University of Bergen, Norway
*Correspondence: jokura@nibb.ac.jp, gaspar.jekely@cos.uni-heidelberg.de

Abstract

text in bold italic underline

Introduction

First descriptions of AO: (Aronova, 1974)

The lobate ctenophore Mnemiopsis

The lithocytes have a large calcium carbonate concretion and… (Tamm, 2014) Living lithocytes are transported along the balancer cilia to the tip of the statocyst (Noda and Tamm, 2014) The projections of a group of bridge cells located on two sides of the apical organ forms an arch and connects the balancers along the tentacular plane (Tamm and Tamm, 2002).

The easiest way is to use the command line to add new refs to the .bib file:

#curl -LH "Accept: application/x-bibtex" https://doi.org/10.7554/eLife.91258.1 >> references.bib

Results

Volume EM reconstruction of the Mnemiopsis apical organ

First, we embedded the entire body of the Mnemiopsis leidyi cydippid-stage larvae, five days post-fertilization, in Epon resin following high-pressure freezing. To reconstruct all synaptic-resolution connections among the cells comprising the apical organ, also known as the aboral organ, we prepared approximately 1,000 ultra-thin serial sections (50 nm thick) from the aboral tip of the larval body embedded in the resin block. Using a scanning electron microscope (Gemini SEM 500) with a resolution of 2.0 nm/pixel, we imaged only the region containing the apical organ. Subsequently, 620 of these images were stitched and aligned using TrakEM2. The resulting volumetric EM dataset had dimensions of 60 μm × 40 μm × 30 μm. We traced and annotated all cells in this dataset, ultimately reconstructing 909 cells, each with both nuclei and cell bodies intact.

Cells containing cilia were traced down to the tips of the cilia, and basal bodies were annotated accordingly. For neuronal skeletonization, nodes were placed to interconnect the profiles of the same neuron’s processes across layers, extending the skeleton until all branches were fully traced. Each node was tagged, and skeletons were named and assigned multi-level annotations. As described later, many neurons we identified formed loop-like structures (anastomosed neurons), wherein separated branches often rejoined either the main trunk or other branches. In such cases, branch nodes were placed near the closest existing node and annotated accordingly. The entire skeletonized volume was composed of 134,591 nodes. However, 88 fragments could not be attached to somata-associated skeletons. Most of these fragments represented short skeletal branches that could not be traced beyond gaps or low-quality layers.

Next, we decided to divide the entire apical organ into four broad quadrants to facilitate grouping the identified cells. The general body plan of ctenophores, when viewed from the aboral side, exhibits biradial symmetry around the positions of the anal pores. This symmetry corresponds to the four blastomeres present at the four-cell stage during early embryonic development. We categorized the traced cells into four quadrants and designated these groups as Q1 through Q4 for clarity and consistency.

Figure 1. Title fig 1 pic of a larva, boxed region of AO, schematic, catmaid pic with slice, dimensions, quadrants, regions, (A) legend (B) legend.

Identification of Synaptic Structures in Syncytium Neurons

Classical neural staining techniques do not provide clear images of the neurons at the aboral pole. However, ultrastructural studies have provided morphological evidence of elements resembling neurons, based on synaptic structures, located on the epithelial floor of the apical organs (Hernandez-Nicaise, 1973; Horridge and Mackay, 1964). Based on these findings, we identified synaptic structures characteristic of ctenophore neurons in our data, using previously identified pre-synaptic triad morphological features, such as single-layered vesicles, smooth endoplasmic reticulum, and tightly packed mitochondria. In our study, we specifically identified synaptic sites and marked mitochondria (orange) as synaptic nodes. The regions where synaptic vesicles align were marked as connectors (light blue arrows) between cells across the membrane. These connectors link the synaptic nodes of the pre-synaptic cytoskeleton to the partner nodes of the post-synaptic cytoskeleton. As previously reported, the specialization of post-synaptic structures in ctenophores is not apparent, so we recognized synaptic vesicle clusters on the pre-synaptic membrane as the point of reference. Synapses were identified as either monoadic or polyadic, with one pre-synaptic neuron forming connections with one or multiple post-synaptic cells.

As mentioned above, following the identification of the presynaptic structures, we reconstructed three major Syncytial neurons. Each of these Syncytial neurons was a cell with multiple nuclei, with membranes fused by continuous plasma membranes. These neurons are distinct in morphology from the Syncytial subepithelial nerve net (SNN) neurons with blebbed morphology previously reconstructed in 3D (Burkhardt_2023?). Our findings represent the second documented discovery of Syncytial neurons in ctenophores. Furthermore, these neurons exhibited clear morphological differences when compared to other sensory cells reported in the same study, such as mesogleal neurons and sensory cells with presynaptic structures and cilia (types 1-4)(Burkhardt_2023?). Based on their distinct spatial relationships, we were able to classify these three Syncytial neurons into two categories. The first type is a larger “AO neuron_Q1234,” which possesses four (or possibly six?) nuclei spanning four quadrants. It contained X presynaptic structures. The second type consists of “AO neuron_Q12” and “AO neuron_Q34,” each having two nuclei spanning two quadrants, and each containing X presynaptic structures.

Figure 2. Title fig 2 pic of a larva, boxed region of AO, schematic, catmaid pic with slice, dimensions, quadrants, regions, (A) legend (B) legend.

Identification the Gravity-Sensitive Neural Circuit via the Syncytial Neurons Network

We discovered that AO neurons form synaptic connections with each other, and that AO neuron_Q1234 forms self-synapses, or “autapses.” To our knowledge, previous reports have not identified synaptic connections between subepithelial nerve net (SNN) neurons. Thus, our results represent the first report of synaptic connectivity between neurons in the apical organ, forming a network in ctenophores (subject to confirmation). These AO neuron networks were found to form many presynaptic structures in relation to the gravity-sensing balancer cells.

Balancer cells are monociliated cells, and their cilia protrude longer than those of other monociliated cells. Moreover, the cilia of balancer cells are bundled into four groups at the center of the apical organ, forming a compound cilium. Based on these features, we identified and classified the balancer cells. The cellular arrangement differed clearly when viewed in the lateral view, sagittal plane, and tentacular plane, with the cell bodies gathering toward the apical organ in the tentacular plane. In each quadrant, the number of cells was as follows: Q1: 37, Q2: 32, Q3: 32, Q4: 28. Each cell contained 3 to 10 ? mitochondria.

From AO neuron_Q1Q2, synapses were formed with 6 of the 37 balancer cells in the Q1 region, and 8 of the 32 balancer cells in the Q2 region. Similarly, from AO neuron_Q3Q4, synapses were formed with 1 of the 32 balancer cells in the Q3 region, and 5 of the 28 balancer cells in the Q4 region. AO neuron_Q1234 formed input synapses with balancer cells in the Q1 region (7 cells), Q2 region (11 cells), Q3 region (6 cells), and Q4 region (10 cells). Some balancer cells received inputs from both AO neuron_Q1Q2 or AO neuron_Q3Q4 and AO neuron_Q1234. While previous studies have suggested the presence of afferent synapses from balancer cells to neurons (Hernandez_Nicaise_1974?), our data did not reveal any synaptic inputs from balancer cells to AO neurons. However, we identified “bridge cells” that actively formed input synapses with AO neurons, highlighting their potential role in the network.

Figure 3. Title fig 3 pic of a larva, boxed region of AO, schematic, catmaid pic with slice, dimensions, quadrants, regions, (A) legend (B) legend.

Identification of Neuron-like Bridge Cells Forming Afferent Synapses in the Gravity-Sensitive Neural Circuit

From our data, we identified several cell groups that form multiple afferent synapses with AO neurons. These cells, based on their morphological features, were found to be the bridge cells first described by Tamm et al. in 2002 (Tamm_and_Tamm_2002?). These cells are characterized by bundles of elongated processes filled with microtubules that arch over the epithelial layer, resembling a bridge. They originate from the base of paired balancer cells along the tentacle surface and extend across the sagittal plane toward the base of the opposite balancer cells. In regions where the mitochondria of the bridge cells are localized (approximately 30%), a presynaptic triad structure, similar to that of AO neurons, was found, containing synaptic vesicles and smooth endoplasmic reticulum.

Our three-dimensional reconstruction data revealed that the bridge cells form two distinct cell groups across the sagittal plane, between the Q1Q2 and Q3Q4 regions. In the Q1Q2 region, 14 cells were identified, while in the Q3Q4 region, 12 cells were identified, totaling 26 bridge cells. Nearly all of these bridge cells (25 out of 26) exhibited afferent synapses from AO neurons. For bridge cells located in the Q1Q2 region, synaptic inputs came primarily from AO neuron_Q1Q2 (11 cells), AO neuron_Q1234 (1 cell), or both (2 cells). For bridge cells located in the Q3Q4 region, synaptic inputs were received from AO neuron_Q3Q4 (1 cell), AO neuron_Q1234 (7 cells), or both (1 cell). In other words, bridge cells in the Q1Q2 region mainly received input from AO neuron_Q1Q2, while those in the Q3Q4 region received input primarily from AO neuron_Q1234, showing a distinct pattern of input in the two regions.

Some bridge cells also formed afferent synapses with AO neurons. For example, bridge cells in the Q1Q2 region formed synapses with AO neuron_Q1Q2 (3 cells) or both AO neuron_Q1Q2 and AO neuron_Q1234 (2 cells). Bridge cells in the Q3Q4 region formed synapses with AO neuron_Q3Q4 (1 cell) or AO neuron_Q1234 (6 cells). A notable difference was observed between the Q1Q2 and Q3Q4 regions in the proportion of synaptic inputs from bridge cells to AO neurons.

Interestingly, in both regions, bridge cells also formed synapses with adjacent bridge cells. However, no synaptic input was found from bridge cells across the sagittal plane to those in the opposite region. To analyze the grouped synaptic connectivity graph, we classified cell types within each region, collapsed cells of the same type into a single node, summed the number of synapses, and explored directed pathways to effectors (balancer cell groups). The results revealed a feedback pathway from AO neurons through the synaptic connections formed by bridge cells, thereby shedding light on the neural circuit structure involving these cells.

Figure 4. Title fig 4 pic of a larva, boxed region of AO, schematic, catmaid pic with slice, dimensions, quadrants, regions, (A) legend (B) legend.

Discussion

Materials and Methods

Acknowledgements

We would like to thank the Jekely lab for the R project template (https://github.com/JekelyLab/new_paper_template) we used to write this paper. This work was funded by … JSPS… ERC.. Others …

References

<<<<<<< HEAD

Sourcing code and working with variable

The ‘analysis/scripts/statistics_for_paper.R’ script is sourced and it runs but the output is not included in the knitted output. But we can access the variables defined in the sourced script simply by adding ` r var_name ` between ` backticks, in this case max_PRC value is (now this number comes from our sourced script).

If we update the data, the script can recalculate the variable we want to refer to in the text and update the number.

References

Aronova M. 1974. Electron microscopic observation of the aboral organ of ctenophora. I. The gravity receptor. Zeitschrift fur mikroskopisch-anatomische Forschung 88:401–412.
Hernandez-Nicaise M-L. 1973. The nervous system of ctenophores III. Ultrastructure of synapses. Journal of Neurocytology 2:249–263. doi:10.1007/bf01104029
Horridge GA, Mackay B. 1964. Neurociliary synapses in pleurobrachia (ctenophora). Journal of Cell Science S3-105:163–174. doi:10.1242/jcs.s3-105.70.163
Noda N, Tamm SL. 2014. Lithocytes are transported along the ciliary surface to build the statolith of ctenophores. Current Biology 24:R951–R952. doi:10.1016/j.cub.2014.08.045
Tamm SL. 2014. Formation of the statolith in the ctenophore mnemiopsis leidyi. The Biological Bulletin 227:7–18. doi:10.1086/bblv227n1p7
Tamm SL, Tamm S. 2002. Novel bridge of axon‐like processes of epithelial cells in the aboral sense organ of ctenophores. Journal of Morphology 254:99–120. doi:10.1002/jmor.10019